The structure and replication of the RNA of Japanese encephalitis virus, a flavivirus, will be studied. Present conflicting data regarding time course of RNA synthesis; number, size and secondary structures of intracellular virus-specific RNA components; effect of cell nucleus inhibitors on RNA replication; and role of cell nucleus in virus production will be experimentally evaluated. The major focus of the program will be isolation of temperature sensitive mutants of the virus, determination of their genetic stability, and their characterization. This will include physical/chemical characteristics of mutant virion populations, ability of the mutants to synthesize virus specific RNA and proteins at the restrictive temperature, and complementation or recombination between pairs of mutants after co-infection of host cells. Related studies will involve defective interfering viruses and persistent viral infections.